The long-term objective of this program is to understand the molecular mechanism of conservative sitespecific recombination by the tyrosine recombinase family of enzymes and to apply this knowledge towards the design of improved tools for the manipulation of DNA molecules. The tyrosine recombinases catalyze strand exchanges between specific DNA sequences to accomplish a variety of important functions in biology, including the integration and excision of phage genomes and the maintenance of circular replicons. The Cre recombinase from bacteriophage P1 has emerged in the last decade to become both a powerful tool for the manipulation of genomes and a paradigm for structure and function in the tyrosine recombinase family. The impact of genomic engineering tools on human health is enormous, given the now widespread use of gene knockout and knock-in experiments, chromosome translocation experiments, and DNA rearrangements in molecular biology procedures. To the extent that an increased mechanistic understanding of these systems can be used to produce improved tools for molecular genetics, the proposed work will also have an important impact on human health. The specific aims of this project are to (i) establish a structural and biochemical basis for understanding the directionality of the Cre-loxP recombination reaction, (ii) determine how Cre mutants and peptide inhibitors are able to support the production, but not the resolution, of the Holliday intermediate of the reaction, (iii) biochemically test specific functional hypotheses based on the Cre-DNA structural models, and (iv) establish a structural framework for understanding site-specific recombination in the more complex and tightly regulated Xer system.